The control 4F6 antibody and the Neuro 2a cell line were included to ensure that the observed result was antigen-specific. The inhibitory effect on EL4 cell viability of both mAbs 8B6 and 14G2a was dose- and time-dependant and became statistically significant at 24 hours post treatment at 20 mg/ml when GDC-0199 compared to mAb 4F6- treated cells . As expected, neither mAb 8B6 nor mAb 14G2a suppressed the growth of the antigen-negative Neuro 2a cell line . Overall, these results show the ability of mAb 8B6 to inhibit tumor cell viability, independently of immunological mechanisms such as CDC and ADCC. To test the ability of both mAbs to induce programmed cell death, we stained antigen-expressing tumor cells with Apo2.7 antibody, followed by flow cytometry analyses, and compared results to control 4F6 antibody-treated cells. Apoptotic cells were detected by flow cytometric analysis after staining bound mAbs to either GD2 or OAcGD2 with a FITC-conjugated goat anti-mouse IgG F 2 fragment goat anti-mouse IgG. This analysis differentiates the antigen expressing cells from the apoptotic one. As shown in Fig. 2B, addition of either mAb 8B6 or mAb 14G2a to the EL4 culture medium resulted in an increased percentage of double-positive cells. Encouragingly, the effects of mAb 8B6 and mAb 14G2a were comparable with about 75% of antigen-positive cells undergoing apoptosis. The above finding was confirmed by fluorescence microscopy of EL4 cells after Hoechst 33342 staining. Microscopic analysis clearly showed bright nuclear staining and highly condensed nuclei with condensed and fragmented chromatin induced by treatment with either mAb 8B6 or mAb 14G2a . These results show the ability of mAb 8B6 to induce apoptosis in OAcGD2�Cexpressing cell lines similarly to mAb 14G2a specific for GD2. The capacity mAb 8B6 or mAb 14G2a to induce CDC and ADCC with EL4 cells in the presence of A-LACK cells and complement from C56BL/6 mice was next evaluated. For CDC assays, the OAcGD2/GD2-expressing target cells were incubated with mAb 14G2a in the presence of diluted mouse serum as complement. Cell death was assessed by the addition of the viability probe propidium iodide.